Supplementary Material to:
Gemcitabine treatment of rat soft tissue sarcoma with
phosphatidyldiglycerol-based thermosensitive liposomes
Simone Limmer1,2, Jasmin Hahn1, Rebecca Schmidt1, Kirsten Wachholz1, Anja
Zengerle1, Katharina Lechner1,2, Hansjörg Eibl3, Rolf D. Issels1,2, Martin Hossann1,2,*,
Lars H. Lindner1,2
1Department
of Internal Medicine III, University Hospital Munich, Ludwig-Maximilians
University, Munich, Germany.
2CCG
Tumor Therapy through Hyperthermia, Helmholtz Zentrum München, German
Research Center for Environmental Health, Munich, Germany.
3Max
Planck Institute for Biophysical Chemistry, Goettingen, Germany.
*Corresponding author: Martin Hossann, Medizinische Klinik und Poliklinik III,
Klinikum der Universität München, Marchioninistrasse 15, D-81377 München,
Germany.
Phone:
++49-89-7095-3153,
Fax:
++49-89-7095-8694.
E-mail:
Martin.Hossann@med.uni-muenchen.de
Quantification of dFdC by high performance liquid chromatography
Gemcitabine (dFdC) was quantified with high performance liquid chromatography
(HPLC) using 5-fluorouridine as internal standard (IS). Representative elution profiles
of samples extracted from human plasma are shown in Supplementary Fig. 1A. Both,
dFdC and IS were separated from extracted plasma components by reversed phase
HPLC using a C18 column (2.6 µm, 100 Å, 100 mm x 3 mm) with 10 mM KH 2PO4 pH
7 as eluent. The Kinetex® column allowed short run times (< 20 min) despite isocratic
flow (0.5 ml/min) at 40°C. Retention times were 5.7 min and 3.7 min for dFdC and IS,
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respectively. Linearity of dFdC (Supplementary Fig. 3B) and IS (data not shown) in
10 mM KH2PO4 were shown from 0 to 2,600 ng. Linearity of dFdC extracted from
spiked plasma samples was observed from 0 to 375 ng (R2 = 0.9958). Approximately
10 ng of dFdC was reliably detectable after extraction from spiked plasma samples
(Supplementary Fig. 3C). Specificity of the method was tested with fetal calf serum
(FCS), human plasma, buffer with empty liposomes and other drugs. No unwanted
peaks in the elution profiles were detected around the retention time of dFdC or IS,
respectively. Recovery from spiked FCS was tested with three concentrations in 6
independent measurements: 35.5 ng/µl, 82.6%; 86.2 ng/µl, 91.0%; 165.0 ng/µl,
90.2%. Precision of the method was tested with five samples contained distinct
DPPG2-TSL preparations encapsulating dFdC. The following mean concentrations
and coefficient of variations (CV) were found (n = 4): 28.8 ng/µl, CV 10.4%; 42.7
ng/µl, CV 6.6%; 59.3 ng/µl, CV 8.7%, 73.8 ng/µl, CV 10.2%; 102.4 ng/µl, CV 11.2%.
The quality of the method was monitored by control charts using the slope of the
linear regression of the calibration curve and mean value of the peak area of IS as
quality characteristics.
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Supplementary Figure 1 - Quantification of dFdC with HPLC. Runs were performed
on C18 columns (2.6 µm, 100 Å, 100 mm x 3 mm) with 10 mM KH 2PO4 pH 7 as
eluent under isocratic flow (0.5 ml/min) at 40°C. Both, dFdC and IS were detected at
275 nm. (A) Overlay of representative elution profiles of dFdC and IS extracted from
spiked plasma samples. (B) Linearity of dFdC in 10 mM KH2PO4 7.0 from 0 to 2,600
ng. (D) Linearity of dFdC from spiked plasma samples from 0 to 93.8 ng (n = 3).
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